Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules

An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Evidence that endogenous 6-(ARG or LYS)-opioid peptides can interact with kappa-receptors as agonists

Evidence is provided for the abilities of endogenous 6-(Arg or Lys)-opioid peptides to interact with kappa-receptors as agonists. Dynorphin-(1-17) and -(1-8), alpha- and beta-neo-endorphin, [Met5]-enkephalin-Arg6-Phe7 and des acetyl salmon endorphin I significantly inhibited the electrically-evoked contractions of rabbit vas deferens which had been shown to contain kappa-receptors exclusively, indicating that endogenous 6-(Arg or Lys)-opioid peptides could act on kappa-receptors as agonists. Additionally, the inhibition of contractions of rabbit vas deferens by 6-(Arg or Lys)-opioid peptides was antagonized more effectively by Mr 2266 which had a high affinity to both mu- and kappa-receptors, than naloxone which had a high affinity only to mu-receptors. This also suggested that 6-(Arg or Lys)-opioid peptides acted as kappa-receptor agonists. The rank order of the inhibitory potency of 6-(Arg or Lys)-opioid peptides against contractions of rabbit vas deferens was as follows: dynorphin-(1-17) greater than alpha-neo-endorphin greater than beta-neo-endorphin .=. dynorphin-(1-8) greater than des acetyl salmon endorphin I greater than [Met5]-enkephalin-Arg6-Phe7. Since other endogenous opioid peptides such as [Met5]- and [Leu5]-enkephalin and beta-endorphin have been shown not to act on kappa-receptors as agonist, data in the present study suggest that endogenous opioid peptides can be classified into two groups in terms of an ability to interact with kappa-receptors as an agonist.     

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Existence of cyclic secondary amino groups in the immunogen

The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-beta-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.     

Met5-enkephalin-Arg6-Gly7-Leu8 immunoreactivity in the human gut

The presence of the proenkephalin A-derived peptide Met5-enkephalin-Arg6-Gly7-Leu8 was demonstrated throughout the human gastrointestinal tract. Highest concentrations of Met5-enkephalin-Arg6-Gly7-Leu8, as assessed by radioimmunoassay, were measured in the separated muscularis externa, while lower levels were found in the submucosa and only small amounts in the mucosa. The results are consistent with a neuronal location of this peptide in the human gut. Over 65% of total immunoreactivity coeluted with the authentic peptide in both molecular exclusion chromatography and HPLC, while most of the remainder activity eluted earlier on gel filtration. The latter material probably represents N-terminally extended Met5-enkephalin-Arg6-Gly7-Leu8. Taken together with previous studies, our results appear to indicate that there are important species differences in post-translational processing of proenkephalin A in gut nerves.     

Identiflcation of a Proenkephalin Precursor in Striatal Tissue

Recent studies have supported the suggestion that proenkephalin is the same in both adrenal medulla and brain. However, although previous investigations have characterized enkephalin-containing adrenal intermediates derived from proenkephalin, as yet no such intermediates have been isolated from the brain. This has led to the belief that the processing of proenkephalin in the brain is extremely rapid and enkephalin-containing intermediates do not accumulate. In this investigation Sephacryl-300 gel filtration chromatography of guinea pig striata, extracted in 8 M urea, demonstrated several peaks of both bioactive and immunoreactive enkephalin-like peptides after enzymatic digest (trypsin followed by carboxypeptidase B). Comparable profiles were obtained using rat and bovine striatal tissue. In guinea pig the major species emerging from gel filtration, eluting with an apparent molecular weight of 29,000, represented approximately 9% of the total (methionine) enkephalin immunoreactivity. It had an apparent pI of 5.0 when subjected to chromatofocusing. This species was further characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and nitrocellulose blotting techniques as well as highly specific radioimmunoassays to (Met5)-enkephalin, (Leu5)-enkephalin, and (Met5)-enkephalin-Arg6-Phe7. This species was found to contain these opioid peptides in an approximately 6:1:1 ratio, respectively, and to have an apparent molecular weight of 31,000. It was also indicated that (Met5)-enkephalin-Arg6-Phe7 constituted the C-terminal seven residues of this molecule.     

Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry

Summary The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro--carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.     

Antibodies to Dopamine: Radioimmunological Study of Specificity in Relation to Immunocytochemistry

Abstract: Two classes of anti-3,4-dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross-linking agent. However, anti-dopamine-glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of antidopamine-glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N -α-acetyl-L-lysine N -methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.     

Evaluation of the immunocytochemical method for amino acids

Free amino acids can be coupled to proteins by glutaraldehyde. Rabbits immunised with a bovine serum albumin-glutaraldehyde-amino acid conjugate form antibodies that recognise similar conjugates with brain proteins in glutaraldehyde-fixed tissue. Antisera raised against conjugated GABA (gamma-aminobutyrate), glutamate, aspartate, taurine, glutamine, or glycine were tested against a variety of small molecular compounds that had been fixed by glutaraldehyde to brain protein and immobilised on cellulose ester filters for processing together with the brain sections. This system permitted closely similar conditions for testing and immunocytochemistry. After removing antibodies against the carrier used for immunisation and against cross reacting amino acid conjugates the antisera showed a high specificity. The specific nature of the antisera was corroborated by solid phase adsorption to the homologous antigens and by inhibition experiments with free amino acids and amino acid-glutaraldehyde fixation complexes. After transection of the striatonigral pathway the ipsilateral substantia nigra was almost depleted of GABA-like immunoreactivity; this observation lends additional support to the selectivity of the GABA antiserum. A semiquantitative relation was established between the concentration of amino acid before fixation in a model system and the subsequent intensity of immunostaining. Similar model experiments suggested that the conjugation of an amino acid to brain protein with glutaraldehyde, and the immunoreactivity of the conjugates, may be significantly inhibited in the presence of high concentrations of other amino compounds.     

Chromatographic characterisation and biological activity of neuropeptides immunoreactive to antisera against Met5-enkephalin-Arg6-Phe7 (YGGFMRF) extracted from the blowfly Calliphora vomitoria (Diptera).

Neuropeptides identified with a radioimmunoassay specific for the C-terminus of Met5-enkephalin-Arg6-Phe7 (YGGFMRF) have been extracted from nervous tissues of the blowfly Calliphora vomitoria and also from whole flies. Chromatographic characterisation, based on criteria of molecular weight, charge and hydrophobicity, reveals a complex multiplicity of immunoreactive peptides. Variations in the amounts and types of peptides found within different nervous tissues is evidence that the cellular precursor processing is selective. Physiological studies on the isolated blowfly salivary gland show that synthetic YGGFMRF is a potent secretagogue with a maximal rate of fluid secretion induced at a concentration of between 10(-13) and 10(-12) M. The tetrapeptide comprising the last four residues of the C-terminus of YGGFMRF, Phe-Met-Arg-Phe, is equally potent. However, the carboxyamidated variants, YGGFMRF-NH2 and the molluscan cardioacceleratory peptide FMRF-NH2, as well as the opioid peptides Met5- and Leu5-enkephalin, have no activity. Partially purified YGGFMRF-immunoreactive peptides from the blowfly have ED50 values in the bioassay approximating to 0.3 thoracic ganglion, 2.1 hypocerebral ganglion and 3.0 brain equivalents.     

Characterization of antibody against human liver guanase by immunoblotting and immunohistochemical staining of human liver guanase by a direct labeling antibody-enzyme method

Human liver guanase was purified and a specific antibody against it was raised in rabbits. The antiserum formed a single precipitin line with human liver extract, and also completely inhibited the activity of the liver enzyme. An immunoblotting study showed that the antibody bound specifically to one band of protein with guanase activity and not to other proteins. Therefore, we concluded that this antiserum against the liver enzyme was suitable for use in immunohistochemical demonstration of guanase. In tissue sections, the immunohistochemical reaction with this antibody was positive in the same locations as the histochemical guanase reaction with DAB (3,3'-diaminobenzidine tetrahydrochloride).     

Reaction of Opioid Peptides with Neutral Endopeptidase (''Enkephalinase")

The kinetics of the reactions of nine opioid peptides with the neutral endopeptidase ("enkephalinase") activities of human kidney, rat kidney, and rat brain have been determined. These opioid peptides can be divided into two classes, those that are good inhibitors of Leu5-enkephalin hydrolysis (Ki less than 75 microM) and good substrates for the enzyme, and those that are poor inhibitors (Ki greater than 500 microM) and are not substrates for the enzyme. The former group includes Leu5-enkephalin, Met5-enkephalin, Met5-enkephalin-Arg6-Phe7, beta-lipotropin, and gamma-endorphin, while the nonreactive opioid peptides include alpha-neo-endorphin, beta-neo-endorphin, dynorphin, and beta-endorphin. These results suggest that those peptides containing the Met5-enkephalin sequence are more reactive than those containing the Leu5-enkephalin sequence. The lack of specificity of this neutral endopeptidase indicates that it may function in the degradation of a variety of biologically active peptides.     

Distribution of Met5-enkephalin-Arg6-Gly7-Leu8-immunoreactivity in the rat and mouse pituitary gland.

The distribution of the octapeptide Met5-enkephalin-Arg6-Gly7-Leu8 (MEAGL), a proenkephalin A-derived opioid peptide, in the rat and mouse pituitary gland was studied using the indirect immunofluorescence technique and immunoelectron microscopy. The anterior lobe contained a few MEAGL-immunoreactive cells but no nerve fibers. A previously unknown enkephalin-immunoreactive nerve fiber system was revealed in the intermediate lobe. These fibers originated in a dense MEAGL-immunoreactive plexus located along the border between the intermediate and posterior lobes and were distributed throughout the lobe. In the posterior lobe, MEAGL immunoreactivity was found in a very dense network of varicose fibers that was evenly distributed over the entire lobe. These results provide a morphological correlate for previous chemical studies and together with them suggest that MEAGL-immunoreactive innervation regulates endocrine functions of the intermediate and posterior lobes directly at the pituitary level.     

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.     

Secretion of [Met]Enkephalyl-Arg6-Phe7-Related Peptides and Catecholamines from Bovine Adrenal Chromaffin Cells: Modification by Changes in Cyclic AMP and by Treatment with Reserpine

Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.     

Characterization of antisera to glutamate and aspartate

Antisera were raised in rabbits against glutamate (Glu) and aspartate (Asp) conjugated to the invertebrate carrier protein hemocyanin (HC) with glutaraldehyde (GA). The antisera were characterized by testing their immunocytochemical staining properties on sections cut at the level of the ventral cochlear nucleus (VCN) from fixed brains of normal rats after absorption with conjugates of compounds structurally similar and biologically relevant to Glu and Asp. Optimal staining with Glu antiserum was obtained at a dilution of 1:10,000 and was completely blocked by 303 micrograms/ml of the Glu-HC conjugate. No crossreactivity with any of 11 compounds tested was observed. Optimal staining with the Asp antiserum was obtained at 1:8000 dilution and was completely blocked by 225 micrograms/ml of the Asp-HC conjugate. Of 10 compounds tested for crossreactivity, only L-asparagine demonstrated a measurable (about 10%) crossreactivity with the Asp antiserum. The specificity of the two antisera was also tested by immunoblot analysis against 11 compounds conjugated to HC with GA. Listed in order of staining intensity, from greatest to least, conjugates that reacted with the Glu antiserum were Glu greater than Gly-Glu greater than Asp-Glu = Asp greater than N-carbamyl (NC)-Glu greater than Asn = Gln = GABA. Conjugates that reacted with the Asp antiserum, in order of decreasing staining intensity, were Asp greater than Glu-Asp = Asn greater than Gly-Asp greater than Glu. No other compounds tested for crossreactivity reacted with the two antisera in the immunoblot analysis. Glu-like immunoreactivity in rat dorsal root ganglia and somatosensory cortex, and the comparative distribution of Glu- and Asp-like immunoreactivities in the latter tissue, are presented as examples of staining patterns obtained with the two antisera.     

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.     

Immunolocalization of CC10 in Clara cells in mouse and human lung

Two antisera, denoted R41 and R42, were raised against a synthetic peptide from the murine Clara cell-specific protein CC10, and one antiserum, denoted R40, was raised against human recombinant uteroglobin, the human homolog of murine CC10. Purified antigen-specific antisera, denoted R40AP, R41AP, and R42AP were prepared using peptide columns. The purified antisera were characterized by dot blots, immunohistochemistry, and immunoblots. Immunohistochemistry of mouse lung showed specific labeling of Clara cells in distal bronchioles by all three antisera. In human lung, the antiuteroglobin antiserum specifically labeled Clara cells, while the anti-mouse peptide antisera had weak crossreactivity and higher background staining. Electron microscopy revealed immunogold labeling of CC10 granules in Clara cells of mouse lung with all antisera. All antisera also labeled a 5-kDa protein on immunoblots of mouse lung homogenates. The surface epithelium of the alveolar air spaces around the distal bronchioles were CC10 positive suggesting a functional activity for CC10 in the lung parenchyma distal to Clara cells. R40AP immunohistochemical staining of sections of normal human lungs and lungs from patients with surfactant protein B deficiency, bronchopneumonia, and idiopathic alveolar proteinosis illustrate the utility of the anti-human CC10 antibody for diagnostic pathology.     

Immuno-affinity purification of specific antibodies against human gastrin releasing peptide (h-GRP) by the h-GRP(1-8)-linked polydimethylacrylamide resin

A new method for immuno-affinity purification of specific antibodies against human gastrin releasing peptide(h-GRP) was developed. The antiserum GP(No. 6201) elicited by h-GRP-BSA conjugate was heterogeneous and reacted not only with h-GRP and its fragments but also partially with other structurally related peptides, such as other GRPs (porcine, canine, and chicken), bombesin, and neuromedin-C. To obtain specific antibodies against human GRP, antiserum GP was purified by column chromatography on the amino-terminal octapeptide h-GRP(1-8)-linked polydimethylacrylamide resin. The antibody thus obtained was highly specific to amino-terminal sequence of h-GRP and hardly reacted with other GRPs (porcine, canine and chicken), bombesin, and even carboxy-terminal h-GRP fragments in ELISA.     

Immunohistochemical localization of carbamoyl-phosphate synthetase (ammonia) in adult rat liver; evidence for a heterogeneous distribution

Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.